LOCUS       sequence-169992-        6055 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  minicircle parental backbone plasmid including luc-pA knock-in donor
            and one cutting site for HITI .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6055)
  AUTHORS   Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, 
            Hatanaka F, Yamamoto M, Araoka T, Li Z, Kurita M, Hishida T, Li M, 
            Aizawa E, Guo S, Chen S, Goebl A, Soligalla RD, Qu J, Jiang T, Fu X,
            Jafari M, Esteban CR, Berggren WT, Lajara J, Nunez-Delicado E, 
            Guillen P, Campistol JM, Matsuzaki F, Liu GH, Magistretti P, Zhang 
            K, Callaway EM, Zhang K, Belmonte JC
  TITLE     In vivo genome editing via CRISPR/Cas9 mediated homology-independent
            targeted integration.
  JOURNAL   Nature. 2016 Dec 1;540(7631):144-149. doi: 10.1038/nature20565. Epub
            2016 Nov 16.
  PUBMED    27851729
REFERENCE   2  (bases 1 to 6055)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nature20565"; journalName: "Nature"; date: "2016-12-1- 1"; 
            volume: "540"; issue: "7631"; pages: "144-149"
COMMENT     Sequence Label: pAi14-luc-MC
FEATURES             Location/Qualifiers
     source          1..6055
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(702..719)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    740..773
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     regulatory      803..812
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             809..2458
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     polyA_signal    2513..2634
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2524..2543)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2578..2597
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3030..3049)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3642..3661
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4078..4095)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4248..4334
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4426..4453
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      4587..5175
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5076..5095
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5329..5346
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5361..5501
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5427..5446)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5587..5609
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 acattaccct gttatcccta gatgacatta ccctgttatc ccagatgaca ttaccctgtt
       61 atccctagat gacattaccc tgttatccct agatgacatt taccctgtta tccctagatg
      121 acattaccct gttatcccag atgacattac cctgttatcc ctagatacat taccctgtta
      181 tcccagatga cataccctgt tatccctaga tgacattacc ctgttatccc agatgacatt
      241 accctgttat ccctagatac attaccctgt tatcccagat gacataccct gttatcccta
      301 gatgacatta ccctgttatc ccagatgaca ttaccctgtt atccctagat acattaccct
      361 gttatcccag atgacatacc ctgttatccc tagatgacat taccctgtta tcccagatga
      421 cattaccctg ttatccctag atacattacc ctgttatccc agatgacata ccctgttatc
      481 cctagatgac attaccctgt tatcccagat gacattaccc tgttatccct agatacatta
      541 ccctgttatc ccagatgaca taccctgtta tccctagatg acattaccct gttatcccag
      601 atgacattac cctgttatcc ctagatacat taccctgtta tcccagatga cataccctgt
      661 tatccctaga tgacattacc ctgttatccc agataaactc aatgatgatg atgatgatgg
      721 tcgagactca gcggccgcgg tgccagggcg tgcccttggg ctccccgggc gcgactatag
      781 aggaacttct tagggcccgc gggccaccat ggaagatgcc aaaaacatta agaagggccc
      841 agcgccattc tacccactcg aagacgggac cgccggcgag cagctgcaca aagccatgaa
      901 gcgctacgcc ctggtgcccg gcaccatcgc ctttaccgac gcacatatcg aggtggacat
      961 tacctacgcc gagtacttcg agatgagcgt tcggctggca gaagctatga agcgctatgg
     1021 gctgaataca aaccatcgga tcgtggtgtg cagcgagaat agcttgcagt tcttcatgcc
     1081 cgtgttgggt gccctgttca tcggtgtggc tgtggcccca gctaacgaca tctacaacga
     1141 gcgcgagctg ctgaacagca tgggcatcag ccagcccacc gtcgtattcg tgagcaagaa
     1201 agggctgcaa aagatcctca acgtgcaaaa gaagctaccg atcatacaaa agatcatcat
     1261 catggatagc aagaccgact accagggctt ccaaagcatg tacaccttcg tgacttccca
     1321 tttgccaccc ggcttcaacg agtacgactt cgtgcccgag agcttcgacc gggacaaaac
     1381 catcgccctg atcatgaaca gtagtggcag taccggattg cccaagggcg tagccctacc
     1441 gcaccgcacc gcttgtgtcc gattcagtca tgcccgcgac cccatcttcg gcaaccagat
     1501 catccccgac accgctatcc tcagcgtggt gccatttcac cacggcttcg gcatgttcac
     1561 cacgctgggc tacttgatct gcggctttcg ggtcgtgctc atgtaccgct tcgaggagga
     1621 gctattcttg cgcagcttgc aagactataa gattcaatct gccctgctgg tgcccacact
     1681 atttagcttc ttcgctaaga gcactctcat cgacaagtac gacctaagca acttgcacga
     1741 gatcgccagc ggcggggcgc cgctcagcaa ggaggtaggt gaggccgtgg ccaaacgctt
     1801 ccacctacca ggcatccgcc agggctacgg cctgacagaa acaaccagcg ccattctgat
     1861 cacccccgaa ggggacgaca agcctggcgc agtaggcaag gtggtgccct tcttcgaggc
     1921 taaggtggtg gacttggaca ccggtaagac actgggtgtg aaccagcgcg gcgagctgtg
     1981 cgtccgtggc cccatgatca tgagcggcta cgttaacaac cccgaggcta caaacgctct
     2041 catcgacaag gacggctggc tgcacagcgg cgacatcgcc tactgggacg aggacgagca
     2101 cttcttcatc gtggaccggc tgaagagcct gatcaaatac aagggctacc aggtagcccc
     2161 agccgaactg gagagcatcc tgctgcaaca ccccaacatc ttcgacgccg gggtcgccgg
     2221 cctgcccgac gacgatgccg gcgagctgcc cgccgcagtc gtcgtgctgg aacacggtaa
     2281 aaccatgacc gagaaggaga tcgtggacta tgtggccagc caggttacaa ccgccaagaa
     2341 gctgcgcggt ggtgttgtgt tcgtggacga ggtgcctaaa ggactgaccg gcaagttgga
     2401 cgcccgcaag atccgcgaga ttctcattaa ggccaagaag ggcggcaaga tcgccgtgaa
     2461 ttcttaactg cagtctagag tcggggcggc cggccgcttc gagcagacat gataagatac
     2521 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     2581 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     2641 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     2701 aagtaaaacc tctacaaatg tggtaaaatc gataaggatc cgtcgaccga tgcccttgag
     2761 agccttcaac ccagtcagct ccttccgggc ccgccccaac tggggtaacc tttgagttct
     2821 ctcagttggg ggtaatcagc atcatgatgt ggtaccacat catgatgctg attataagaa
     2881 tgcggccgcc acactctagt ggatctcgag ttaataattc agaagaactc gtcaagaagg
     2941 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg
     3001 tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga
     3061 tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc
     3121 accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc
     3181 atgctcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc
     3241 agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt
     3301 ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca
     3361 tcagccatga tggatacttt ctcggcagga gcaaggtgta gatgacatgg agatcctgcc
     3421 ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag
     3481 ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcttgcagtt
     3541 cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca
     3601 gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata
     3661 gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa
     3721 acgatcctca tcctgtctct tgatcagagc ttgatcccct gcgccatcag atccttggcg
     3781 gcgagaaagc catccagttt actttgcagg gcttcccaac cttaccagag ggcgccccag
     3841 ctggcaattc cggttcgctt gctgtccata aaaccgccca gtctagctat cgccatgtaa
     3901 gcccactgca agctacctgc tttctctttg cgcttgcgtt ttcccttgtc cagatagccc
     3961 agtagctgac attcatccgg ggtcagcacc gtttctgcgg actggctttc tacgtgctcg
     4021 aggggggcca aacggtctcc agcttggctg ttttggcgga tgagagaaga ttttcagcct
     4081 gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag
     4141 tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga
     4201 tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa
     4261 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
     4321 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt
     4381 ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga
     4441 cggatggcct ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat
     4501 atgtatccgc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4561 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4621 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4681 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4741 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4801 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4861 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4921 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4981 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5041 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5101 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5161 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5221 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     5281 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     5341 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5401 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     5461 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     5521 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     5581 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     5641 agcagatcaa ttcgcgcgcg aaggcgaagc ggcatgcata atgtgcctgt caaatggacg
     5701 aagcagggat tctgcaaacc ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta
     5761 ccaattatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact
     5821 cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa
     5881 aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg
     5941 cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga
     6001 aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgat
//
